Bottom Line:
We found that DJ-1 was expressed early during zebrafish development and throughout adulthood.While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos.Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

ABSTRACTMutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

Figure 7: Lack of changes in DJ-1 expression in the presenceof AÎ². Conditioned media from CHO and CHO+APP cells were collected for the measurement of AÎ² levels and treatment of PC-12 cells. A. High levels of AÎ²40 were found in the media from CHO+APP cells before (white bar) and after (black bar) the treatment of PC-12 cells (the standard error of means was illustrated). B. High levels of AÎ²42 were found in the media from CHO+APP cells before (white) and after (black) the treatment of PC-12 cells, compared to undetectable amount of AÎ² in the media from CHO cells. C. Conditioned media from CHO or CHO+APP cells were applied to PC-12 cells for 24 hr, and cells were collected for the quantification of DJ-1 by Western blot using antibody DJ-1-N. Cells from two independent experiments were collected, and the expression levels of DJ-1 in both sets of PC-12 cells maintained at similar levels.

Mentions:
To search for any mechanistic cause for DJ-1 expression in AD brains, we examined the effect of AÎ² on the regulation of DJ-1 expression. We have used two cell lines, CHO that expresses endogenous levels of APP, and CHO+APP that expresses high levels of APP and generates a large amount of AÎ². When we measured the conditioned media from CHO and CHO+APP cells, we found elevated levels of AÎ²40 (Fig. 7A, before treatment) and AÎ²42 (Fig. 7B, before treatment) in the media from CHO+APP cells, compared to undetectable level of AÎ² in the media from CHO cells (Fig. 7A and 7B). We applied conditioned media to PC-12 cells for 24 hrs, and collected media and cells for the quantification of AÎ² and DJ-1. We found that the conditioned media continued to carry high levels of AÎ²40 (Fig. 7A, after treatment) and AÎ²42 (Fig. 7B, after treatment). Despite of the AÎ² exposure, the expression levels of DJ-1 in PC-12 cells maintained at similar levels (Fig. 7C). Therefore, endogenous DJ-1 expression in PC-12 cells did not change in the presence of extracellular AÎ².

Figure 7: Lack of changes in DJ-1 expression in the presenceof AÎ². Conditioned media from CHO and CHO+APP cells were collected for the measurement of AÎ² levels and treatment of PC-12 cells. A. High levels of AÎ²40 were found in the media from CHO+APP cells before (white bar) and after (black bar) the treatment of PC-12 cells (the standard error of means was illustrated). B. High levels of AÎ²42 were found in the media from CHO+APP cells before (white) and after (black) the treatment of PC-12 cells, compared to undetectable amount of AÎ² in the media from CHO cells. C. Conditioned media from CHO or CHO+APP cells were applied to PC-12 cells for 24 hr, and cells were collected for the quantification of DJ-1 by Western blot using antibody DJ-1-N. Cells from two independent experiments were collected, and the expression levels of DJ-1 in both sets of PC-12 cells maintained at similar levels.

Mentions:
To search for any mechanistic cause for DJ-1 expression in AD brains, we examined the effect of AÎ² on the regulation of DJ-1 expression. We have used two cell lines, CHO that expresses endogenous levels of APP, and CHO+APP that expresses high levels of APP and generates a large amount of AÎ². When we measured the conditioned media from CHO and CHO+APP cells, we found elevated levels of AÎ²40 (Fig. 7A, before treatment) and AÎ²42 (Fig. 7B, before treatment) in the media from CHO+APP cells, compared to undetectable level of AÎ² in the media from CHO cells (Fig. 7A and 7B). We applied conditioned media to PC-12 cells for 24 hrs, and collected media and cells for the quantification of AÎ² and DJ-1. We found that the conditioned media continued to carry high levels of AÎ²40 (Fig. 7A, after treatment) and AÎ²42 (Fig. 7B, after treatment). Despite of the AÎ² exposure, the expression levels of DJ-1 in PC-12 cells maintained at similar levels (Fig. 7C). Therefore, endogenous DJ-1 expression in PC-12 cells did not change in the presence of extracellular AÎ².

Bottom Line:
We found that DJ-1 was expressed early during zebrafish development and throughout adulthood.While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos.Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

ABSTRACTMutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.